Three hundred and fifty million people are infected by the hepatitis B Virus (HBV) and 300 million people by Hepatitis C Virus (HCV), worldwide.These viral infections are characterized by a high risk of evolution toward chronic infection (5% for HBV in adult life, and 60-80% in the neonatal life; 70-80% for HCV). Chronic infection by HBV and/or HCV may evolve to cirrhosis (10-20%) and to liver cancer (5% of cirrhoses evolve to HCC per year). Antiviral treatments, including Interferon, Lamivudine for HBV and combination of Interferon and Ribavirin for HCV, have a limited efficacy since they determine long-term responses in around 30-40% of patients.

Liver carcinogenesis is related to chronic liver inflammation due to HBV and/or HCV, to the direct effects of viral proteins and, for HBV, to the integration of the HBV genome into the cellular genome of liver cells. 1) Chronic inflammation plays a major role by the modification of the control of processes like liver fibrosis and liver cell proliferation. Actually, 90% of liver cancers develop on a cirrhotic liver and 30-50% of patients with cirrhosis develop a liver cancer over a period of 10 years. 2) Certain HBV and HCV viral proteins, as well as the integration of the HBV DNA into the cellular DNA can affect the control of liver cell proliferation and apoptosis and the response of liver cells to interferon and to cytokines (TNFalpha, etc ); 3) recurrent genetic abnormalities (deletions and amplifications of certain chromosomal loci) have been identified thanks to genetic studies performed in HCCs (reviewed in:Ozturk Seminars in liver diseases;1999,19:235. Bréchot:Seminars in Cancer biology, 2000;10:211-231, Buendia: Viral Hepatitis; Zuckerman et Thomas Ed, Churchill Livingstone 1998, 179-200;Thorgeirsson SS, Grisham JW, Nature Genetics,31,339,2002).

In this context, the research program of our Unit is focused on the following issues :

Liver cell adhesion and proliferation :

Research team C. Desdouets. This team studies the mechanisms regulating the liver cell ploidy, during liver development and during pathological conditions like viral infections and liver carcinogenesis. This study involves: the quantification of the DNA of liver cell nuclei, the study of proteins implicated in the cytodieresis and of kinases inhibitors (P27 and P21). In 2003, this team has characterised the in vivo kinetic of liver polyploidisation and has demonstrated that the inhibition of cytokinesis determines the formation of binucleated hepatocytes, a major step in the process of polyploidisation.

Research team L. Christa. This team has shownthe interaction of a type C lectin with the sub-unit RIIa of PKA. The ongoing project studies the in vivo function of the HIP-PAP protein, in embryo and liver stem cells and during liver cell regeneration in transgenic mice, by looking for protein-protein interactions (protein chip Ciphergen method).

Research team J. Faivre. This teamhas demonstratednew splicing variants of the Cyclin A2 gene. Studies on cell localisation and function of the corresponding proteins are ongoing.

Persistence of hepatitis B and hepatitis C viruses :

1. Research team D. Kremsdorf. a) This team studies the MxA protein and has confirmed its antiviral effect in vivo. Transgenic mice for HBV and the MxA gene have been crossed and the expression level of HBV has been studied. In double transgenic female mice the team has observed a decrease of HBV replication. This demonstrates the implication of the MxA protein in the antiviral effect of interferon against HBV ; b) The team works on the in vivo expression of the HBSP protein, encoded by one of the spliced HBV transcripts and found that it correlates with the viral replication as well as with the severity of liver fibrosis. c) It has been recently reported that immunodeficient mice transgenic for the urokinase plasminogen activator can be engrafted with human hepatocytes.The team combined these findings with previous indications that depletion of non-adaptative defenses was critical to the survival of heterologous grafts. Depletion of non adaptative defenses dramatically improved the survival of human hepatocytes. Furthermore, the highly differentiated status of transplanted human hepatocytes was confirmed by their receptivity to Plasmodium falciparum in the liver stage development. This provides a new tool to study in vivo the biology of infection of hepatotropic viruses.

2. Research team V. Thiers. This team is studying the expression profile of microdissected liver cells with steatosis. Liver needle biopsy specimens from patients with chronic HCV infection, with and without steatosis, are under investigation...

Liver carcinogenesis and viral infections (HBV, HCV) :

Research team N. Pavio, C. Bréchot: 1. This team studies the biological effect of core sequences isolated from the tumorous and non tumorous tissue of patients with liver cancer. It has shown that core proteins, derived from tumor, target the TGF-b pathway, involved in fibrosis and anti-proliferative effects, suggesting that this pathway might be impaired in infected cells and favor cell proliferation and tumorigesis. The precise molecular mechanism of this inhibition is under investigation.

Research team V. Thiers. This team has studied, in microdissecetd liver cells, the amount of HCV RNA obtained from the tumorous and non tumorous tissue and has shown that the amount of viral RNA does not correlate with the degree of liver damage. Another project of this team is to characterize full core sequences isolated from microdissected hepatocytes in tumour and non-tumour counterpart tissue from the liver of HCV-infected patients. The impact (if any) of amino acid variations on the secondary structure of the protein will be studied using bioinformatic tools. The phenotype effects of the mutations identified in vivo will be also examined.

Research team V. Thiers. The integration of laser-assisted microdissection and proteomic analysis could identify novel protein markers useful for diagnosis and prognosis. This study will combine microdissection with two-dimensional electrophoresis (2DE). Protein fingerprinting of matched pairs of HCC and non-HCC tissues will be compared and the differentially expressed proteins identified by mass spectrometry (MALDI-TOF-MS).

Research team P. Paterlini-Brechot. This team has shown that the HBV genome integrates into a cellular gene in more than 50% of HBV related HCCs. The genes targeted by HBV DNA integration are key genes in cell signalling pathways (MAPK, calcium signalling genes, transcription factors etc..). Moreover, recurrent integrations have been found in the telomerase gene and in the IP3R (inositol tri-phosphate receptor gene). This result demonstrates for the first time the impact of insertional mutagenesis in a human cancer and shows its key role in liver carcinogenesis.

Research team P. Paterlini-Brechot. This team has shown the impact of calcium signalling in the biological effect of the X protein of HBV and of the core protein of HCV, two proteins which have been involved in the process of liver carcinogenesis. This result opens a new research domain aiming to study the impact of calcium signalling in liver carcinogensis.

New approaches for the treatment of liver cancer

1. Research team J. Faivre. Radioiodine (131I) therapy is a recognized, well-tolerated approach in the treatment of human thyroid cancer, thanks to the ability of thyroidal cells to trap and retain iodine through the activity of a specific protein, the Natrium Iodide Symporter (NIS). The goal of this team is to define an efficient specific procedure of viral vector-mediated NIS gene delivery in the liver followed by 131I radiotherapy in order to destroy the tumoral hepatocytes. Using an adenoviral CMV-NIS vector, the team found strong and sustained NIS-related radioiodine uptake in tumor-affected livers of DEN rats, and a strong inhibition of tumor growth, the complete regression of small nodules, and prolonged survival in this aggressive model of HCC.
2. Research team P. Paterlini-Brechot. This team has shown the impact of detection and counting, by the ISET technique, circulating tumor cells (CTC) in patients with liver cancer. In fact, the study has shown that the presence and number of CTC significantly correlates with a reduced survival in 44 patients with HCC followed for a mean period of one year. CTC have also been individually microdissected and molecular analyses have been performed to look for beta-catenin mutations (which are found in around 20% of HCCs). The results show that beta-catenin mutations are rarely found in CTC (3 out of 60) demonstrating that this mutation is not required for the first step of tumor cells invasion.

ISET has also been applied to isolate circulating fetal cells from mothers at risk of having a baby with spinal amiotrophy. This study has shown that ISET may be considered a promising approach to develop a new approach of non invasive prenatal diagnosis.

Clinical research projects :

Our Unit has set up a bank of DNA isolated from patients infected by HCV, and patients with alcoholic cirrhosis to search for genetic polymorphism associated with liver diseases. Several pharmacological studies are ongoing targeting the treatment of HBV and HCV chronic infections. The clinical teams are studying the impact of the HCV infection on lipid metabolism and its relationship with viremia and anti-viral treatments. They also study the evolution of patients with biliary cholestatic liver diseases and the effect of the treatments on these diseases. A project dealing with the role of genetic susceptibility in the development and/or the severity of PBC is in process.

Two new teams have integrated the Unit 370 in 2003, working on HBV-HCV immuno-pathology.

Team A. Budkowska. This team has recently shown that HCV core protein and HCV nucleocapsids have the capacity to bind non-immune IgG via their Fcgregions.. The FcgR-like site of the core protein is conformational, and requires the presence of amino acid sequence (3-75) for optimal activity. Several human viruses (such CMV, HSV-1 a VZV and EBV) encode receptors that bind the IgG Fcg domain non-immune IgG. Virus proteins with functional properties of FcgRs may enable the virus to evade immune surveillance by avoiding the effector consequences of antibody binding. The FcgR-like function of the core protein, expressed on HCV nucleocapsids or on infected cells, may offer a survival advantage to HCV by interfering with immunological defence mechanisms mediated by the Fcg region of anti-core antibodies, and with functions of FcRn.

Team M L Michel. This team hasdeveloped HLA-A2/HBsAg double transgenic mice. This humanized animal model allows to design new strategies aimed at restoring in vivo effector functions of peripherally tolerized T cells, that could be directly transferred to patients chronically infected with HBV. It has provided evidences for the immunological efficacy of HBV-DNA vaccination and demonstrated that DNA vaccination can restore T cell responses in chronic HBV-carriers. This group uses HBsAg as virus like particle (VLP) for the presentation of foreign epitopes derived from SIV and HIV to specifically activate the immune system. This approach was combined to genetic vaccination in order to provide T cell help and to enhance cytotoxic response to these viruses. In a pilot experiment in macaques, it appeared that priming with hybrid DNA greatly enhanced virus-specific T cell responses when animals were boosted with recombinant poxviruses (MVA), and provided partial protection from SHIV89.6P mucosal challenge.